Gene Therapy for Lysosomal storage diseases
Lysosomal storage diseases (LSDs) encompass over 40 distinct inherited metabolic disorders (1). Most LSDs are classified by a lack of or a significant decrease in the activity of a lysosomal enzyme. A sub class of LSDs are caused by deficiencies in non-enzymatic proteins which can be involved in activation of lysosomal enzymes or are involved in the intracellular trafficking of proteins to the lysosome (2). In both cases molecules are not properly degraded by the lysosome which leads to their accumulation within the cell (Figure 1, diagrams LSDs). Their build up then leads to progressive cellular, tissue, and organ dysfunction (3). LSD phenotypes vary depending on what lysosomal associated protein is effected and to what extent it can function (3). An example of an LSD is Pompe disease which results from a deficiency in the lysosomal enzyme maltase. This enzyme breaks down glycogen into glucose. The lack of maltase activity results in the accumulation of glycogen which leads to dysfunction. This results in cardiomyopathy as well as skeletal and respiratory muscle weakness (1). LSDs, like Pompe disease, are generally the result of mutations in single genes making. LSDs a good candidate for gene therapy. Current Treatments for Lysosomal Storage Disorders Current treatments of LSDs include enzyme replacement (ER) and haematopoietic stem cell transplantation (HSCT). In ER, functioning enzyme is administered to the patent to replace the deficient enzyme. Once in the blood stream the enzyme will bind to either the mannose-6-phosphate receptor (M6PR) which is ubiquitously expressed or to the mannose receptor (ManR) which is only expressed by cells of th e reticuloendothelial system upon which it the receptor is endocytosed allowing the enzyme to entire the cell (2) (Figure 2) . Once in the cell the enzyme can be trafficked to the lysosome. ER has been shown to be effect in treating several LSDs including Pompe disease, Gaucher disease, and Fabry disease (3). However these enzymes are unable to pass the blood brain barrier thus making ER ineffective at treating central nervous system LSDs (2). This is a major drawback as approximately 75% of LSDs effect the central nervous system (CNS) (4). In HSCT, stem cells taken from bone marrow or umbilical cord blood are transferred to the patient (4). These cells can produce lysosomal proteins, some of which will be released by the cell and will then enter the blood stream allowing them to be circulated through the body (4). These enzymes enter cells by binding to either M6PR or ManR followed by endocytosis (2). The process of lysosomal proteins from one cell being taken up by another is known as "cross-correction" (5) (Figure 2). The goal of HSCT is to provide a continuous and wide spread source of the deficient protein (4). Unlike ER, HSCT has been shown to be able to treat CNS LSDs, as the haematopoietic stem cells can enter the CNS and differentiate into microglia (6). HSCT has been used to treat infantile Krabbe disease, adrenoleukodystrophy, and metachomatic leukodystrophy (3). Some major drawbacks of HSCT include harsh regiments for patients and graft versus host disease (4). HSCT and ER can improve the quality of life as well as prolong it but cannot cure LSDs (4). HSCT and ER are only effective in treating a small fraction of LSDs thus gene therapy could expand the therapeutic options available to treat these disorders. Gene Therapy and Lysosomal Storage Disorder LSDs are good candidates for gene therapy for several reasons. Most LSDs are the result of single gene defects making replacement of gene not overly difficult. Lysosomal proteins are ubiquitously expressed so there is no concern in genetically altering the wrong cell type. Over expression of lysosomal proteins has not been shown to be detrimental thus precise control of gene expression is not required. Gene therapy can also take advantage of "cross correction" meaning that the number of cells that need to be genetically altered is relatively low (4). Recombinant viral gene transfer is the most effective form of gene therapy for LSDs (4). This involves using viruses to deliver therapeutic genes (7). Several viral vectors have been used for treatment of LSDs. These include γ-retroviruses, herpes simplex virus (HSV), adenovirus, adeno-associated virus (AAV), and lenti-virus (4). AAV and lenti-virus show the most promise for treating LSDs. Lenti-viruses can transfect both dividing and non-dividing cells and have low immunogenicity (4). Previous research has shown that human cells transduced using lentiviruses and then transplanted into mice were successful in treating LSDs (8). A recent study (9) coupled HSCT with gene therapy. In this study the researchers isolated haematopoietic stem cells from 3 patients who were suffering from the LSD metachromatic leukodystrophy, which is caused by a deficiency in the enzyme arylsulfatase A and primarily effects the CNS, and transduced these cells using lentiviruses carrying a gene that encodes a functional arylsulfatase A. The patients were monitored for 24 months and showed no signs of disease progression (9) suggesting that this is an effective method for treating CNS LSDs in humans (Figure 3). AAVs are another common vector used for gene therapy. AAVs are capable of highly efficient and stable transduction of liver, skeletal muscle, and heart cells (3). Direct injection of AAVs has also been shown to lead to highly efficient transduction of neurons (10). AAVs are capable of expression transgenes in immune competent animal models however they do elicit immune responses (11) and existing immunity can impair transduction (3). Research has demonstrated that AAVs can be used to treat LSDs that manifest in the CNS as well as in liver and muscle tissue (12,13). Direct injection of AAV carrying the gene encoding for human acid sphingomyelinase, an enzyme deficient in Niemen-Pick disease, into the thalaums and brain stem of crab-eating macaques resulted in successful transduction as measured by enzyme levels (14). These data suggest that AAVs could be used to treat LSDs in humans. Both AAV and lenti-viral methods of gene delivery are promising and could be developed further to treat LSDs. Potential Problems There are potential problems associated with the use of lenti-viruses and AAVs. While lenti-viruses do not elicit immune responses (15) they may induce epigenetic modifications in transduced cells (16). This may result in significantly altered cell functioning. While AAVs have been shown to elicit mild immune responses in mice (4). 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